Esophageal cancer is the 6th leading cause of cancer related deaths in American males with nearly 16,000 cases each year. Prognosis of this cancer remains poor with a five-year survival rate of 17%, indicating a need for further investigation int the molecular mechanisms underlying invasion and metastases. Mutations in TP53 are found in over 40% of esophageal cancer cases. While p53 has primarily been considered to be a tumor suppressor in its wild-type state through its role in trans-activating genes that respond to cellulr stresses, recent findings have suggested that mutant p53 may act through neomorphic mechanisms to promote tumor progression. Our prior work utilized overexpression of EGFR and DNA-binding domain (DBD) mutations in TP53 to transform immortalized primary esophageal cells. These transformed cells can invade in 3D- organotypic (3D OTC) culture to varying degrees, depending upon the type of TP53 mutation. Additionally, laser capture microdissection (LCM) with microarray analysis, comparing invasive versus non-invasive cells, showed preferential dysregulation in genes associated with a migratory phenotype in the invasive cells. We hypothesize that the invasive gene signature of these transformed esophageal epithelial cells is mediated by mutant p53 and its gain-of-function properties. This hypothesis will be tested through the three interrelated Specific Aims. Aim I: To determine the role of mutant p53 as an aberrant transcription factor. This will be achieved by expression of a p53 triple mutant, which maintains its mutant conformation but loses its trans-activation capability, in 2D and 3D systems, to determine if it can rescue the mutant p53 phenotype in vitro. We will assay for proliferation, migration, invasion, and anchorage-independent growth in 2D, and morphology in 3D OTC. Additionally, we will perform a ChIP-seq in the transformed esophageal cell lines with mutant p53 to determine novel targets of mutant p53. Aim 2: To investigate mutant p53/p63 protein-protein interaction and its contribution to the invasive gene signature. Loss of Np63 has been associated with increase in metastases and poor survival in esophageal squamous cell carcinoma (ESCC). Moreover, mutant p53 has been shown to interact with deltaNp63. We will characterize this protein-protein interaction in vitro in our transformed cells by assaying for dirct interaction and determine if this interaction inhibits downstream deltaNp63 functions on target genes through luciferase reporter assays. In Aim 3, we will generate a mouse model of ESCC, to confirm the role of the downstream targets in mutant p53- mediated invasion. We will drive overexpression of a constitutively active EGFR and mutant p53, under an L2-promoter, which is specific for oral squamous, esophageal, and forestomach tissues. After aging these transgenic mice, we will examine the invasive and metastatic tumors for expression of the target genes, determined in Aims 1 and 2. Furthermore, we will characterize these mice for tumor development and progression. New insights will be acquired into the functional roles of mutant p53 in tumor invasion.